Base-modified enzymatic nucleic acid

ABSTRACT

Method to produce a more active ribozyme by introducing a modified base into a substrate binding arm of the ribozyme or its catalytic core.

[0001] This application is a continuation-in-part of McSwiggen,“Optimization of Ribozyme Activity”, U.S. Ser. No. 07/963,322, filedOct. 15, 1992 and Usman et al., entitled “Base-modified enzymaticnucleic acid”, U.S. Ser. No. 08/149,210 (filed Nov. 8, 1993), the wholeof both of which, including drawings, is hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to enzymatic RNA molecules or ribozymeshaving a modified nucleotide base sequence.

[0003] Ribozymes are RNA molecules having an enzymatic activity which isable to repeatedly cleave other separate RNA molecules in a nucleotidebase sequence specific manner. Such enzymatic RNA molecules can betargeted to virtually any RNA transcript, and efficient cleavageachieved in vitro. Kim et al., 84 Proc. Nati. Acad. Sci. USA 8788, 1987;Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988;and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.

[0004] Ribozymes act by first binding to a target RNA. Such bindingoccurs through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA which acts tocleave the target RNA. Thus, the ribozyme first recognizes and thenbinds a target RNA through complementary base-pairing, and once bound tothe correct site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

[0005] By “complementarity” is meant a nucleic acid that can formhydrogen bond(s) with other RNA sequence by either traditionalWatson-Crick or other non-traditional types (for example, Hoogsteentype) of base-paired interactions.

[0006] Six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. Table I summarizes some of the characteristicsof these ribozymes. In general, enzymatic nucleic acids act by firstbinding to a target RNA. Such binding occurs through the target bindingportion of a enzymatic nucleic acid which is held in close proximity toan enzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

[0007] The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the effective concentration of ribozyme necessary to effect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of binding, but also on the mechanismby which the molecule inhibits the expression of the RNA to which itbinds. That is, the inhibition is caused by cleavage of the RNA targetand so specificity is defined as the ratio of the rate of cleavage ofthe targeted RNA over the rate of cleavage of non-targeted RNA. Thiscleavage mechanism is dependent upon factors additional to thoseinvolved in base pairing. Thus, it is thought that the specificity ofaction of a ribozyme is greater than that of antisense oligonucleotidebinding the same RNA site.

[0008] The following discussion of relevant art is dependent on thediagram shown in FIG. 1, in which the numbering of various nucleotidesin a hammerhead ribozyme is provided. This is not to be taken as anindication that the Figure is prior art to the pending claims, or thatthe art discussed is prior art to those claims.

[0009] Odai et al., FEBS 1990, 267:150, state that substitution ofguanosine (G) at position 5 of a hammerhead ribozyme for inosine greatlyreduces catalytic activity, suggesting “the importance of the 2-aminogroup of this guanosine for catalytic activity.”

[0010] Fu and McLaughlin, Proc. Natl. Acad. Sci. (USA) 1992, 89:3985,state that deletion of the 2-amino group of the guanosine at position 5of a hammerhead ribozyme, or deletion of either of the 2′-hydroxylgroups at position 5 or 8, resulted in ribozymes having a decrease incleavage efficiency.

[0011] Fu and McLaughlin, Biochemistry 1992, 31:10941, state thatsubstitution of 7-deazaadenosine for adenosine residues in a hammerheadribozyme 6an cause reduction in cleavage efficiency. They state that the“results suggest that the N⁷-nitrogen of the adenosine (A) at position 6in the hammerhead ribozyme/substrate complex is critical for efficientcleavage activity.” They go on to indicate that there are five criticalfunctional groups located within the tetrameric sequence GAUG in thehammerhead ribozyme.

[0012] Slim and Gait, 1992, BBRC 183, 605, state that the substitutionof guanosine at position 12, in the core of a hammerhead ribozyme, withinosine inactivates the ribozyme.

[0013] Tuschl et al., 1993 Biochemistry 32, 11658, state thatsubstitution of guanosine residues at positions 5, 8 and 12, in thecatalytic core of a hammerhead, with inosine, 2-aminopurine, xanthosine,isoguanosine or deoxyguanosine cause significant reduction in thecatalytic efficiency of a hammerhead ribozyme.

[0014] Fu et al., 1993 Biochemistry 32, 10629, state that deletion ofguanine N⁷, guanine N² or the adenine N⁶-nitrogen within the core of ahammerhead ribozyme causes significant reduction in the catalyticefficiency of a hammerhead ribozyme.

[0015] Grasby et al., 1993 Nucleic Acids Res. 21, 4444, state thatsubstitution of guanosine at positions 5, 8 and 12 positions within thecore of a hammerhead ribozyme with O⁶-methylguanosine results in anapproximately 75-fold reduction in k_(cat).

[0016] Seela et al., 1993 Helvetica Chimica Acta 76, 1809, state thatsubstitution of adenine at positions 13, 14 and 15, within the core of ahammerhead ribozyme, with 7-deazaadenosine does not significantlydecrease the catalytic efficiency of a hammerhead ribozyme.

[0017] Adams et al., 1994 Tetrahedron Letters 35, 765, state thatsubstitution of uracil at position 17 within the hammerheadribozyme•substrate complex with 4-thiouridine results in a reduction inthe catalytic efficiency of the ribozyme by 50 percent.

[0018] Ng et al., 1994 Biochemistry 33, 12119, state that substitutionof adenine at positions 6, 9 and 13 within the catalytic core of ahammerhead ribozyme with isoguanosine, significantly decreases thecatalytic activity of the ribozyme.

[0019] Jennings et al., U.S. Pat. No. 5,298,612, indicate thatnucleotides within a “minizyme” can be modified. They state-

[0020] “Nucleotides comprise a base, sugar and a monophosphate group.Accordingly, nucleotide derivatives or modifications may be made at thelevel of the base, sugar or monophosphate groupings . . . . . Bases maybe substituted with various groups, such as halogen, hydroxy, amine,alkyl, azido, nitro, phenyl and the like.”

SUMMARY OF THE INVENTION

[0021] This invention relates to production of enzymatic RNA moleculesor ribozymes having enhanced or reduced binding affinity and enhancedenzymatic activity for their target nucleic acid substrate by inclusionof one or more modified nucleotides in the substrate binding portion ofa ribozyme such as a hammerhead, hairpin, VS ribozyme or hepatitis deltavirus derived ribozyme. Applicant has recognized that only small changesin the extent of base-pairing or hydrogen bonding between the ribozymeand substrate can have significant effect on the enzymatic activity ofthe ribozyme on that substrate. Thus, applicant has recognized that asubtle alteration in the extent of hydrogen bonding along a substratebinding arm of a ribozyme can be used to improve the ribozyme activitycompared to an unaltered ribozyme containing no such altered nucleotide.Thus, for example, a guanosine base may be replaced with an inosine toproduce a weaker interaction between a ribozyme and its substrate, or auracil may be replaced with a bromouracil (BrU) to increase the hydrogenbonding interaction with an adenosine. Other examples of alterations ofthe four standard ribonucleotide bases are shown in FIGS. 6a-d withweaker or stronger hydrogen bonding abilities shown in each figure.

[0022] In addition, applicant has determined that base modificationwithin some catalytic core nucleotides maintains or enhances enzymaticactivity compared to an unmodified molecule. Such nucleotides are notedin FIG. 7. Specifically, referring to FIG. 7, the preferred sequence ofa hammerhead ribozyme in a 5′ to 3′ direction of the catalytic core isCUG ANG A G•C GAA A, wherein N can be any base or may lack a base(abasic); G•C is a base-pair. The nature of the base-paired stem II(FIGS. 1, 2 and 7) and the recognition arms of stems I and III arevariable. In this invention, the use of base-modified nucleotides inthose regions that maintain or enhance the catalytic activity and/or thenuclease resistance of the hammerhead ribozyme are described. (Baseswhich can be modified include those shown in capital letters).

[0023] Examples of base-substitutions useful in this invention are shownin FIGS. 6, 8-14. In preferred embodiments cytidine residues aresubstituted with 5-alkylcytidines (e.g., 5-methylcytidine, FIG. 8,R═CH₃, 9), and uridine residues with 5-alkyluridines (e.g.,ribothymidine (FIG. 8, R═CH₃, 4) or 5-halouridine (e.g., 5-bromouridine,FIG. 8, X═Br, 13) or 6-azapyrimidines (FIGS. 8, 17) or 6-alkyluridine(FIG. 14). Guanosine or adenosine residues may be replaced bydiaminopurine residues (FIG. 8, 22) in either the core or stems. Inthose bases where none of the functional groups are important in thecomplexing of magnesium or other functions of a ribozyme, they areoptionally replaced with a purine ribonucleoside (FIGS. 8, 23), whichsignificantly reduces the complexity of chemical synthesis of ribozymes,as no base-protecting group is required during chemical incorporation ofthe purine nucleus. Furthermore, as discussed above, base-modifiednucleotides may be used to enhance the specificity or strength ofbinding of the recognition arms with similar modifications.Base-modified nucleotides, in general, may also be used to enhance thenuclease resistance of the catalytic nucleic acids in which they areincorporated. These modifications within the hammerhead ribozyme motifare meant to be non-limiting example. Those skilled in the art willrecognize that other ribozyme motifs with similar modifications can bereadily synthesized and are within the scope of this invention.

[0024] Substitutions of sugar moieties as described in the art citedabove, may also be made to enhance catalytic activity and/or nucleasestability.

[0025] Thus, in a first aspect, the invention features a modifiedribozyme having one or more substrate binding arms including one or moremodified nucleotide bases; and in a related aspect, the inventionfeatures a method for production of a more active modified ribozyme(compared to an unmodified ribozyme) by inclusion of one or more of suchmodified nucleotide bases in a substrate binding arm.

[0026] The invention provides ribozymes having increased enzymaticactivity in vitro and in vivo as can be measured by standard kineticassays. Thus, the kinetic features of the ribozyme are enhanced byselection of appropriate modified bases in the substrate binding arms.Applicant recognizes that while strong binding to a substrate by aribozyme enhances specificity, it may also prevent separation of theribozyme from the cleaved substrate. Thus, applicant provides means bywhich optimization of the base pairing can be achieved. Specifically,the invention features ribozymes with modified bases with enzymaticactivity at least 1.5 fold (preferably 2 or 3 fold) or greater than theunmodified corresponding ribozyme. The invention also features a methodfor optimizing the kinetic activity of a ribozyme by introduction ofmodified bases into a ribozyme and screening for those with higherenzymatic activity. Such selection may be in vitro or in vivo. Byenchanced activity is meant to include activity measured in vivo wherethe activity is a reflection of both catalytic activity and ribozymestability. In this invention, the product of these properties inincreased or not significantly (less that 10 fold) decreased in vivocompared to an all RNA ribozyme.

[0027] By “enzymatic portion” is meant that part of the ribozymeessential for cleavage of an RNA substrate.

[0028] By “substrate binding arm” is meant that portion of a ribozymewhich is complementary to (i.e., able to base-pair with) a portion ofits substrate. Generally, such complementarity is 100%, but can be lessif desired. For example, as few as 10 bases out of 14 may bebase-paired. Such arms are shown generally in FIGS. 1-3 as discussedbelow. That is, these arms contain sequences within a ribozyme which areintended to bring ribozyme and target RNA together through complementarybase-pairing interactions; e.g., ribozyme sequences within stems I andIII of a standard hammerhead ribozyme make up the substrate-bindingdomain (see FIG. 1).

[0029] By “unmodified nucleotide base” is meant one of the basesadenine, cytosine, guanosine, uracil joined to the 1′ carbon ofβ-D-ribo-furanose. The sugar also has a phosphate bound to the 5′carbon. These nucleotides are bound by a phosphodiester between the 3′carbon of one nucleotide and the 5′ carbon of the next nucleotide toform RNA.

[0030] By “modified nucleotide base” is meant any nucleotide base whichcontains a modification in the chemical structure of an unmodifiednucleotide base which has an effect on the ability of that base tohydrogen bond with its normal complementary base, either by increasingthe strength of the hydrogen bonding or by decreasing it (e.g., asexemplified above for inosine and bromouracil). Other examples ofmodified bases include those shown in FIGS. 6a-d and other modificationswell known in the art, including heterocyclic derivatives and the like.

[0031] In preferred embodiments the modified ribozyme is a hammerhead,hairpin VS ribozyme or hepatitis delta virus derived ribozyme, and thehammerhead ribozyme includes between 32 and 40 nucleotide bases. Theselection of modified bases is most preferably chosen to enhance theenzymatic activity (as observed in standard kinetic assays designed tomeasure the kinetics of cleavage) of the selected ribozyme, i.e., toenhance the rate or extent of cleavage of a substrate by the ribozyme,compared to a ribozyme having an identical nucleotide base sequencewithout any modified base.

[0032] By “enzymatic nucleic acid molecule” it is meant a nucleic acidmolecule which has complementarity in a substrate binding region to aspecified gene target, and also has an enzymatic activity which isactive to specifically cleave RNA in that target. That is, the enzymaticnnucleic acid molecule is able to inter-molecularly cleave RNA andthereby inactivate a target RNA molecule. This complementarity functionsto allow sufficient hybridization of the enzymatic nucleic acid moleculeto the target RNA to allow the cleavage to occur. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% mayalso be useful in this invention.

[0033] In preferred embodiments of this invention, the enzymatic nucleicacid molecule is formed in a hammerhead or hairpin motif, but may alsobe formed in the motif of a hepatitis delta virus, group I intron orRNaseP RNA (in association with an RNA guide sequence) or Neurospora VSRNA. Examples of such hammerhead motifs are described by Rossi et al.,1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs byHampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929,and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example ofthe hepatitis delta virus motif is described by Perrotta and Been, 1992Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al.,U.S. Pat. No. 4,987,071. These specific motifs are not limiting in theinvention and those skilled in the art will recognize that all that isimportant in an enzymatic nucleic acid molecule of this invention isthat it has a specific substrate binding site which is complementary toone or more of the target gene RNA regions, and that it have nucleotidesequences within or surrounding that substrate binding site which impartan RNA cleaving activity to the molecule.

[0034] In a preferred embodiment the invention provides a method forproducing a class of enzymatic cleaving agents which exhibit a highdegree of specificity for the RNA of a desired target. The enzymaticnucleic acid molecule is preferably targeted to a highly conservedsequence region of a target RNAs such that specific treatment of adisease or condition can be provided with either one or severalenzymatic nucleic acids. Such enzymatic nucleic acid molecules can bedelivered exogenously to specific cells as required.

[0035] By “kinetic assays” or “kinetics of cleavage” is meant anexperiment in which the rate of cleavage of target RNA is determined.Often a series of assays are performed in which the concentrations ofeither ribozyme or substrate are varied from one assay to the next inorder to determine the influence of that parameter on the rate ofcleavage.

[0036] By “rate of cleavage” is meant a measure of the amount of targetRNA cleaved as a function of time.

[0037] In a second aspect, enzymatic nucleic acid having a hammerheadconfiguration and modified bases which maintain or enhance enzymaticactivity is provided. Such nucleic acid is also generally more resistantto nucleases than unmodified nucleic acid. By “modified bases” in thisaspect is meant those shown in FIG. 6 A-D, and 8, or their equivalents;such bases may be used within the catalytic core of the enzyme as wellas in the substrate-binding regions. As noted above, substitution in thecore may decrease in vitro activity but enhances stability. Thus, invivo the activity may not be significantly lowered. As exemplifiedherein such ribozymes are useful in vivo even if active over all isreduced 10 fold. Such ribozymes herein are said to “maintain” theenzymatic activity on all RNA ribozyme.

[0038] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The drawings will first briefly be described.

[0040] Drawings

[0041]FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be ≧2 base-pair long. Each N isindependently any base or non-nucleotide as used herein.

[0042]FIG. 2a is a diagrammatic representation of the hammerheadribozyme domain known in the art; FIG. 2b is a diagrammaticrepresentation of the hammerhead ribozyme as divided by Uhlenbeck (1987,Nature, 327, 596-600) into a substrate and enzyme portion; FIG. 2c is asimilar diagram showing the hammerhead divided by Haseloff and Gerlach(1988, Nature, 334, 585-591) into two portions; and FIG. 2d is a similardiagram showing the hammerhead divided by Jeffries and Symons (1989,Nucl. Acids. Res., 17, 1371-1371) into two portions.

[0043]FIG. 3 is a diagrammatic representation of the general structureof a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 basepairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally providedof length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20or more). Helix 2 and helix 5 may be covalently linked by one or morebases (i.e., r is≧1 base). Helix 1, 4 or 5 may also be extended by 2 ormore base pairs (e.g., 4-20 base pairs) to stabilize the ribozymestructure, and preferably is a protein binding site. In each instance,each N and N′ independently is any normal or modified base and each dashrepresents a potential base-pairing interaction. These nucleotides maybe modified at the sugar, base or phosphate. Complete base-pairing isnot required in the helices, but is preferred. Helix 1 and 4 can be ofany size (i.e., o and p is each independently from 0 to any number,e.g., 20) as long as some base-pairing is maintained Essential bases areshown as specific bases in the structure, but those in the art willrecognize that one or more may be modified chemically (abasic, base,sugar and/or phosphate modifications) or replaced with another basewithout significant effect. Helix 4 can be formed from two separatemolecules, i.e., without a connecting loop. The connecting loop whenpresent may be a ribonucleotide with or without modifications to itsbase, sugar or phosphate. “q” is ≧2 bases. The connecting loop can alsobe replaced with a non-nucleotide linker molecule. H, refers to bases A,U or C. Y refers to pyrimidine bases.

[0044]FIG. 4 is a representation of the general structure of thehepatitis delta virus ribozyme domain known in the art.

[0045]FIG. 5 is a representation of the general structure of theself-cleaving VS RNA ribozyme domain.

[0046]FIGS. 6a-d are diagrammatic representations of standard basemodifications for adenine, guanine, cytosine and uracil, respectively.

[0047]FIG. 7 is a diagrammatic representation of a position numberedhammerhead ribozyme (according to Hertel et al., Nucleic Acids Res.1992, 20:3252) showing specific substitutions in the catalytic core andsubstrate binding arms. Compounds 4, 9, 13, 17, 22, 23 are described inFIG. 8.

[0048]FIG. 8 is a diagrammatic representation of various nucleotidesthat can be substituted in the catalytic core of a hammerhead ribozyme.

[0049]FIG. 9 is a diagrammatic representation of the synthesis of aribothymidine phosphoramidite.

[0050]FIG. 10 is a diagrammatic representation of the synthesis of a5-methylcytidine phosphoramidite.

[0051]FIG. 11 is a diagrammatic representation of the synthesis of5-bromouridine phosphoramidite.

[0052]FIG. 12 is a diagrammatic representation of the synthesis of6-azauridine phosphoramidite.,

[0053]FIG. 13 is a diagrammatic representation of the synthesis of2,6-diaminopurine phosphoramidite.

[0054]FIG. 14 is a diagrammatic representation of the synthesis of a6-methyluridine phosphoramidite.

[0055]FIG. 15 is a representation of a hammerhead ribozyme targeted tosite A (HHA). Site of 6-methyl U substitution is indicated.

[0056]FIG. 16 shows RNA cleavage reaction catalyzed by HHA ribozymecontaining 6-methyl U-substitution (6-methyl-U4). U4, represents a HHAribozyme containing no 6-methyl-U substitution.

[0057]FIG. 17 is a representation of a hammerhead ribozyme targeted tosite B (HHB). Sites of 6-methyl U substitution are indicated.

[0058]FIG. 18 shows RNA cleavage reaction catalyzed by HHB ribozymecontaining 6-methyl U-substitutions at U4 and U7 positions(6-methyl-U4). U4, represents a HHB ribozyme containing no 6-methyl-Usubstitution.

[0059]FIG. 19 is a representation of a hammerhead ribozyme targeted tosite C (HHC). Sites of 6-methyl U substitution are indicated.

[0060]FIG. 20 shows RNA cleavage reaction catalyzed by HHC ribozymecontaining 6-methyl U-substitutions at U4 and U7 positions(6-methyl-U4). U4, represents a HHC ribozyme containing no 6-methyl-Usubstitution.

[0061]FIG. 21 shows 6-methyl-U-substituted HHA ribozyme-mediatedinhibition of rat smooth muscle cell proliferation.

[0062]FIG. 22 shows 6-methyl-U-substituted HHC ribozyme-mediatedinhibition of stromelysin protein production in human synovialfibroblast cells.

[0063] Modified Ribozymes

[0064] There is a narrow range of binding free-energies between aribozyme and its substrate that will produce maximal ribozyme activity.Such binding energy can be optimized by making ribozymes with G to I andU to BrU substitutions (or equivalent substitutions) in thesubstrate-binding arms. This allows manipulation of the bindingfree-energy without actually changing the target recognition sequence,the length of the two substrate-binding arms, or the enzymatic portionof the ribozyme. The shape of the free-energy vs. ribozyme activitycurve can be readily determined using data from experiments in whicheach base (or several bases) is modified or unmodified, and without thecomplication of changing the size of the ribozyme/substrate interaction.

[0065] Such experiments will indicate the most active ribozymestructure. It is likely that only one or two modifications are necessarysince a very small change in binding free energy (even one base-pairinteraction) can dramatically affect ribozyme activity; the use ofmodified bases thus permits “fine tuning” of the binding free energy toassure maximal ribozyme activity. In addition, replacement of suchbases, e.g., I for G, may permit a higher level of substrate specificitywhen cleavage of non-target RNA is a problem.

[0066] Method

[0067] Modified substrate binding arms can be: synthesized usingstandard methodology. For example, phosphoramidites of inosine and5-bromouracil can be used. Generally, a target site that has beenoptimized for stem I and III lengths (in a hammerhead ribozyme—otherribozymes can be treated in a similar manner), and that has G and/or Uin the ribozyme portion of stem I and III, is selected. Modifiedribozymes are made by replacing various G and U residues with I and BrU,respectively, during synthesis of the ribozyme. The modified ribozymesare then tested to determine kinetic parameters using standardprocedures (see McSwiggen, “Improved Ribozymes”, U.S. Ser. No.07/884,521, filed May 14, 1992, hereby incorporated by referenceherein). The binding affinities for the ribozymes can also be determinedby standard procedures, e.g., by T-melt, gel-binding, or by competitionkinetics assays. By comparison of binding affinity and ribozyme activitythe optimum binding affinity of a ribozyme can then be found. Othercombinations of G, I, U BrU, and other bases can then be tested withnearly identical binding free energy, but different base sequence, todetermine whether factors other than simple binding free-energy play arole.

[0068] It is preferred to perform routine experiments of this type toselect a desired ribozyme substrate binding sequence by use of anunmodified ribozyme with a modified substrate (which contains themodified bases). That is, the reverse experiment to that described aboveis performed. Such an experiment is more readily performed since thesubstrate is generally shorter than the ribozyme, and can be readilysynthesized without concern about its secondary structure. Thus, asingle ribozyme can be tested against a plurality of modified substratesin order to define which of the substrates provides better kineticresults. Once a preferred substrate is identified, the ribozyme can thenbe modified in a way which mirrors the selected substrate, and thentested against an unmodified substrate.

[0069] Such experiments will define useful ribozymes of this inventionin which one or more modified bases are provided in the substratebinding arms with greater enzymatic activity in vitro and in vivo thancomparable unmodified ribozymes. Such modifications may also beadvantageous if they increase the resistance of a ribozyme to enzymaticdegradation in vivo.

[0070] Synthesis of Ribozymes

[0071] Synthesis of nucleic acids greater than 100 nucleotides in lengthis difficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small enzymatic nucleicacid motifs (e.g., of the hammerhead or the hairpin structure) are usedfor exogenous delivery. The simple structure of these moleculesincreases the ability of the enzymatic nucleic acid to invade targetedregions of the mRNA structure.

[0072] The ribozymes are chemically synthesized. The method of synthesisused follows the procedure for normal RNA synthesis as described inUsman et al., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al.,1990 Nucleic Acids Res., 18, 5433 and makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. The average stepwise coupling yieldswere >98%.

[0073] Ribozymes are purified by gel electrophoresis using generalmethods or are purified by high pressure liquid chromatography (HPLC;See Usman et al., Synthesis, deprotection, analysis and purification ofRNA and ribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 thetotality of which is hereby incorporated herein by reference) and areresuspended in water.

[0074] Various modifications to ribozyme structure can be made toenhance the utility of ribozymes. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch ribozymes to the target site, e.g., to enhance penetration ofcellular membranes, and confer the ability to recognize and bind totargeted cells.

[0075] Optimizing Ribozyme Activity

[0076] Ribozyme activity can be optimized as described by Stinchcomb etal., “Method and Composition for Treatment of Restenosis and CancerUsing Ribozymes,” filed May 18, 1994, U.S. Ser. No. 08/245,466. Thedetails will not be repeated here, but include altering the length ofthe ribozyme binding arms (stems I and III, see FIG. 2c), or chemicallysynthesizing ribozymes with modifications that prevent their degradationby serum ribonucleases (see e.g., Eckstein et al., InternationalPublication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565;Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trendsin Biochem. Sci. 17, 334; Usman et al., International Publication No. WO93/15187; and Rossi et al., International Publication No. WO 91/03162,as well as Usman, N. et al. U.S. Patent Application 07/829,729, andSproat, European Patent Application 92110298.4 which describe variouschemical modifications that can be made to the sugar moieties ofenzymatic RNA molecules. Modifications which enhance their efficacy incells, and removal of stem II bases to shorten RNA synthesis times andreduce chemical requirements. (All these publications are herebyincorporated by reference herein.).

[0077] Administration of Ribozyme

[0078] Sullivan et al., PCT WO94/02595, describes the general methodsfor delivery of enzymatic RNA molecules . Ribozymes m ay be administeredto cells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres. For some indications, ribozymes may be directly deliveredex vivo to cells or tissues with or without the aforementioned vehicles.Alternatively, the RNA/vehicle combination is locally delivered bydirect injection or by use of a catheter, infusion pump or stent. Otherroutes of delivery include, but are not limited to, intravascular,intramuscular, subcutaneous or joint injection, aerosol inhalation, oral(tablet or pill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan et al., supra and Draper etal., PCT WO 93/23569 which have been incorporated by reference herein.

EXAMPLES

[0079] The following are non-limiting examples showing the synthesis ofbase-modified catalytic nucleic acids.

Example 1

[0080] Synthesis of Hammerhead Ribozymes Containing Base-ModifiedNucleotides

[0081] The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al., J. Am. Chem. Soc. 1987, 109,7845-7854 and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441and makes use of common nucleic acid protecting and coupling groups,such as dimethoxytrityl at the 5′-end, and phosphoramidites at the3′-end (compounds 4, 9, 13, 17, 22, 23). The average stepwise couplingyields were >98%. These base-modified nucleotides may be incorporatednot only into hammerhead ribozymes, but also into hairpin, VS ribozymes,hepatitis delta virus, or Group 1 or Group 2 introns. They are,therefore, of general use as replacement motifs in any nucleic acidstructure.

[0082] In the case of the hammerhead ribozyme the following specificsubstitutions may be used:

[0083] Referring to FIG. 7, in the catalytic core (numberednucleotides), the pyrimidine C3 may be replaced by the cytosine analogsshown in FIG. 6c and compound 9 in FIG. 8.

[0084] Referring to FIG. 7, in the catalytic core (numberednucleotides), the pyrimidines U4 and N7 may be replaced by the cytosineanalogs shown in FIG. 4c, the uridine analogs shown in FIG. 6d andcompounds 4, 9, 13 and 17 in FIG. 8 and FIG. 14.

[0085] Referring to FIG. 7, in the catalytic core (numberednucleotides), the purines G5, G8 and G12 may be replaced by the guanineanalogs shown in FIG. 6b and compounds 22 and 23 in FIG. 8.

[0086] Referring to FIG. 7, in the catalytic core (numberednucleotides), the purines A6, A9, A13 and A14 may be replaced by theadenine analogs shown in FIG. 6a and compounds 22 and 23 in FIG. 8.

[0087] Referring to FIGS. 1 and 5, in stems I, II and III any of thepyrimidines may be replaced by the pyrimidine analogs shown in FIGS. 6cand 6d and compounds 4, 9, 13 and 17 in FIG. 8 and FIG. 14 as long asbase-pairing is maintained in the stems.

[0088] Referring to FIGS. 1 2 and 7, in stems I, II and III any of thepurines may be replaced by the purine analogs shown in FIGS. 6a and 6band compounds 22 and 23 in FIG. 8 as long as base-pairing is maintainedin the stems.

[0089] Referring to FIGS. 1 2 and 7, in loop II (denoted as loop II inFIG. 7) any nucleotide may be replaced by the pyrimidine analogs shownin FIGS. 6c and 6d, the purine analogs shown in FIGS. 6a and 6 b andcompounds 4, 9, 13, 17, 22 and 23 in FIG. 8 and FIG. 14.

Example 2

[0090] Synthesis of Ribothymidine Phosphoramidite 4

[0091] Referring to FIG. 9, ribothymidine 1 was prepared according toVorbrüggen et al., Chem. Ber. 1981, 114:1234, and tritylated to yieldDMT derivative 2. 2 was silylated to yield 2′-O-TBDMS derivative 3. Thephosphoramidite 4 was prepared according to Scaringe et al., NucleicAcids Res. 1990, 18:5433.

Example 3

[0092] Synthesis of 5-Methylcytidine Phosphoramidite 9

[0093] Referring to FIG. 10, Ribothymidine 1 (4 g, 15.5 mmol) wascoevaporated with dry pyridine (2 x 100 ml) and redissolved in drypyridine (100 ml). To the resulting solution 4,4′-DMT-Cl (6.3 g, 18.6mmol) was added and the reaction mixture was left at room temperature(about 20-25° C. for 16 hours. The reaction mixture was quenched withmethanol (25 ml) and evaporated to dryness. The residue was partitionedbetween chloroform and 5% sodium bicarbonate. The organic layer waswashed with 5% sodium bicarbonate and brine, then dried over sodiumsulfate and evaporated. The residue was additionally dried bycoevaporation with dry pyridine (2 x 50 ml) then redissolved in drypyridine (100 ml) and acetic anhydride (4.4 ml, 46.5 mmol) was added tothe resulting solution. The reaction mixture was left at roomtemperature overnight, then quenched with methanol (25 ml), evaporatedand worked-up as outlined above. The crude5′-O-dimethoxytrityl-2′,3′,-di-O-acetyl-ribo-thymidine 5 was purified byflash chromatography on silica gel,(hexanes:ethylacetate:triethylamine/45:45:10 to give 6.86 g (68.7%) of 5as a yellowish foam.

[0094] Triethylamine (14.72 ml, 105.6 mmol) was added dropwise to astirred ice-cooled mixture of triazole (6.56 g, 95.04 mmol) andphosphorous oxychloride (2 ml, 21.2 mmol) in 100 ml of dry acetonitrile.A solution of nucleoside 5 (6.89, 10.56 mmol) in 50 ml of dryacetonitrile was added dropwise to the resulting suspension and thereaction mixture was stirred at room temperature for 4 hours. Thereaction was concentrated, dissolved in chloroform and washed with asaturated aqueous solution of sodium bicarbonate, water, dried oversodium sulfate and evaporated to dryness. To a solution of the residue(7.24 g) in dioxane (120 ml) was added 40 ml of 29% aqueous NH₄OH andthe resulting solution was left overnight, then evaporated to dryness toyield 6.86 g of crude cytidine derivative 6 which was used withoutpurification.

[0095] To a solution of 6 (3.5 g, 6.25 mmol) in dry pyridine (100 ml)was added 3.97 ml of trimethylchlorosilane to transiently protect freesugar hydroxyls. The reaction mixture was then treated with isobutyrylchloride (0.98 ml, 9.375 mmol) for 5 hours. The resulting mixture wasquenched with 10 ml of methanol, then 10 ml of water was added and after10 minutes 10 ml of 29% aq. ammonia was added and the reaction mixturewas stirred for 2 hours and evaporated to dryness. The resulting residuewas worked-up as outlined above for the compound 5 and purified by flashchromatography on silica gel (ethylacetate:hexanes/1:3) to yield 2.37 g(60%) of the nucleoside 7.

[0096] To a solution of compound 7 (1.3 g, 2.06 mmol) in dry pyridine0.97 g (5.72 mmol) of silver nitrate-was added followed by 2.86 ml of a1 M solution of tert-butyidimethyl chloride in THF. The reaction mixturewas left for 8 hours, evaporated, and dissolved in chloroform. Thesilver salt precipitate was filtered off and the reaction solution waswashed with 5% aq. sodium bicarbonate and brine, dried over sodiumsulfate and evaporated. The mixture of 2′- and 3′-isomers was separatedby flash chromatography on silica gel (hexanes:ethylacetate/4:1) toyield 0.62 g (40%) of 2′-isomer 8, which was converted to thephosphoramidite 9 by the general method described in Scaringe et al.,Nucleic Acids Res. 1990, 18:5433.

Example 4

[0097] Synthesis of 5-Bromouridine Phosphoramidite 13 (See, Talbat etal., Nucl. Acids Res. 18:3521-21, 1990)

[0098] Referring to FIG. 11, 5-Bromouridine 10 (1.615 g, 5 mmol) wascoevaporated with dry pyridine and redissolved in dry pyridine. To theresulting solution 2.03 g (6 mmol) of DMT-Cl was added and the reactionmixture was left overnight. After work-up and purification by flashchromatography on silica gel (chloroform:methanol/95:5) 2.5 g (80%) ofthe dimethoxytritylated compound 11 was obtained.

[0099] To a solution of 11 (2 g) in dry pyridine was added 1.5 eq. ofTBDMS-Cl for 2 days. The reaction mixture was evaporated, dissolved inchloroform, washed with 5% aq. sodium bicarbonate and brine. The organiclayer was dried over sodium sulfate, evaporated and purified by flashchromatography on silica gel (ethylacetate:hexanes/1:2) to yield 1.4 g(60%) of 2′-isomer 12, which was converted to the phosphoramidite 13 bythe general method described in Scaringe et al., Nucleic Acids Res.1990, 18:5433.

Example 5

[0100] Synthesis of 6-azauridine Phosphoramidite 17

[0101] Referring to FIG. 12, 6-Azauridine (4.9 g, 20 mmol) wasevaporated with dry pyridine (2 x 100 ml) and dissolved in dry pyridine(100 ml) and after addition of 4,4′-DMT-Cl (7.45 g, 22 mmol) left for 16hours at room temperature. The reaction mixture was diluted with dryMeOH (50 ml), evaporated to dryness, coevaporated with toluene (2x 100ml), the residue dissolved in CHCl₃ (500 ml) and washed with 5% NaHCO₃(100 ml), brine (100 ml), dried, and purified by flash chromatography (agradient CHCl₃ to 5% EtOH/CHCl₃ to yield 1 g (92.2%) of intermediate)15.

[0102] To a solution of 15 (3.23 g, 5.9 mmol) in 100 ml of dry THF,AgNO₃ (7.08 mmol) and dry pyridine (2.1 ml, 4.4 mmol) were added. Thereaction mixture was stirred at room temperature until full dissolutionof AgNO₃ (about 1 hour) occurred. Then 7 ml of a 1 M solution ofTBDMS-Cl in THF was added and the reaction mixture stirred for 16 hoursat room temperature. The reaction mixture was filtered and the filtrateevaporated to dryness. The resulting residue was dissolved in CHCl₃ (300ml) and washed with 5% NaHCO₃ (100 ml), brine (100 ml), dried, andpurified by flash chromatography (gradient of hexanes to hexanes:ethylacetate/1:1) to yield 3.71 g (62%) of 2′-TBDMS-isomer 16 which wasconverted to the phosphoramidite 17 by the general method described inScaringe et al., Nucleic Acids Res. 1990, 18:5433.

Example 6

[0103] Synthesis of 2,6-diaminopurine Phosphoramidite 22

[0104] Referring to FIG. 13, phosphoramidite 22 was prepared by thegeneral method described in Scaringe et al., Nucleic Acids Res. 1990,18:5433. Specifically, guanosine (11.32 g, 40 mmol) was dried bycoevaporation with dry pyridine and redissolved in dry pyridine.Chlorotrimethylsilane (26.4 ml, 208 mmol) was added under stirring tothe above solution and the reaction mixture was stirred overnight. Tothe resulting persilylated guanosine derivative phenylacetylchloride(12.7 ml, 96 mmol) was added dropwise and the reaction mixture wasstirred for 12 hours. The reaction was quenched with 50 ml of methanoland 50 ml of water and stirred for 15 minutes, then 50 ml of 29% ammoniawas added and the reaction mixture left for an additional 2 hours.Solvents were removed in vacuo, and the resulting oil was partitionedbetween ethyl acetate and water. The separated water layer was washedwith ethyl acetate and was precipitated at 4° C. The resulting solid wasfiltered off to give 8 g of N²phenylacetylguanosine 18. The motherliquor was concentrated to give additional crop (4 g). Overall yield˜12g (75%).

[0105] N²Phenylacetylguanosine 18 (2.3 g, 5.73 mmol) was dried bycoevaporation (3 times) with dry pyridine and dissolved in 50 ml of drypyridine. To the resulting solution dimethoxytritylohloride (2.33 g,6.88 mmol) was added and the reaction mixture was left at roomtemperature for 5 hours. The reaction was quenched with 25 ml ofmethanol and evaporated to dryness. The residue was dissolved indichloromethane, washed with 5% aq. sodium bicarbonate and brine, driedover sodium sulfate and evaporated. The resulting oil was further driedby coevaporation with dry pyridine, dissolved in pyridine and treatedwith acetic anhydride (1.4 ml) for 4 hours at room temperature. Thereaction mixture was quenched and worked-up as described above. Thecrude final compound was purified by flash chromatography on silica gelusing dichloromethane:methanol/98:2 mixture as eluent. The desiredfractions were collected and evaporated to give 3.5 g (77%) of5′-O-dimethoxytrityl-2′,3′-di-O-acetyl-N²-phenylacetylguanosin e 19 as ayellowish foam.

[0106] To a solution of compound 19 (3.5 g, 4.45 mmol) in 50 ml of drydichloromethane, containing 3.11 ml of dilsopropylethylamine, was addedmesitylenesulfonyl chloride (1.9 g, 8.9 mmol) and dimethylaminopyridine(0.28 g). The reaction mixture was stirred for 30 minutes, evaporatedand purified by flash chromatography on silica gel using dichloromethane(11) followed by 2% Methanol in dichloromethane (0.71) to give 2.8 g(64%) of O⁶-mesitylene intermediate 20. To a solution of 20 in 40 ml ofdry tetrahydrofuran lithium disulfide (0.3 g, 6.8 mmol) was added andthe reaction mixture was stirred for 20 hours. The resulting clearsolution was evaporated and worked-up as described above. The residuewas purified by flash chromatography on silica gel in 1% Methanol indichloromethane to give 1.1 g (31%) of5′-O-dimethoxytrityl-2′,3′-di-O-acetyl-N2phenylacetyl-6-thiogua nosine21.

[0107] To an ice-cooled (0° C.) solution of5′-O-dimethoxytrityl-2′,3′-di-O-acetyl-N²phenylacetyl-6-thiogua nosine21 (1 g) in pyridine:methanol/20 ml:2.6 ml, 2.4 ml of 1 M aq. sodiumhydroxide were added and the reaction mixture was allowed to stay at 0°C. for 20 minutes. The solution was neutralized with Dowex 2x8 (Pyr⁺) topH 7. The resin was filtered off and washed with aq. pyridine. Thecombined filtrate and washings were evaporated and dried in vacuo togive quantitatively 5′-O-dimethoxytrityl-N²phenylacetyl-6-thioguanosine.

[0108] To a stirred suspension of5′-O-dimethoxytrityl-N²phenylacetyl-6-thioguanosine (1.13 g, 1.57 mmol)in dry acetonitrile (35 ml) and triethylamine (1 ml) was addeddinitrofluorobenzene (0.34 g, 1.88 mmol) and the reaction mixture wasstirred under anhydrous conditions for 2 hours. The reaction wasevaporated and worked-up as described for compound 20 and purified byflash chromatography on silica gel in 1% methanol in chloroform(containing 1% triethylamine) as an eluent to give 0.93 g (67%) of5′-O-dimethoxytrityl-N²phenylacetyl-6-S-dinitrophenyl guanosine.

[0109] To a solution of5′-O-dimethoxytrityl-N²phenylacetyl-6-S-dinitrophenyl guanosine (0.9 g,1 mmol) in dry pyridine t-butyldimethylsilylchloride (0.46 g, 3 mmol)and tetrabutylammonium nitrate (3 mmol) were added and the reactionmixture was left for 50 hours. TLC (hexane:ethyl acetate/3:1) showeddisappearance of the starting material and formation of two newcompounds with a predominance of a lower R_(f) (3′-O-silyl isomeraccording to 1H-NMR). The desired 2′-isomer (70 mg) was obtained afterevaporation and work-up and separation by flash chromatography on silicagel using hexane:ethyl acetate/4:1 as eluent. The remaining mixture wasrearranged in methanol with 2 drops of triethylamine and separated asabove. This rearrangement procedure was repeated twice to finally give250 mg of the desired 2′-isomer.5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyl-N²phenylacetyl-6-S-dinitrophenylguanosine.

[0110] 5′-O-Dimethoxytrityl-2′-O-t-butyidimethylsilyl-N²phenylacetyl-6-S-dinitrophenyl guanosine (0.18 g, 0.18 mmol) was dissolved in drytetrahydrofuran under dry argon. N-Methylimidazole (0.01 ml, 0.09 mmol)and sym-collidine (0.178 ml, 1.35 mmol) were added and the solution wasice-cooled. 2-Cyanoethyl N,N′-diisopropylchlorophosphoramidite (0.083ml, 0.36 mmol) was added dropwise and stirring was continued for 3 hoursat room temperature. The reaction mixture was again ice-cooled andquenched with 6 ml of dry degassed ethyl acetate. After 5 min stirringthe mixture was concentrated in vacuo (40° C.), dissolved in chloroform,washed with 5% aq sodium bicarbonate, then with brine and evaporated.The residue was purified by flash chromatography on silica gel usingethyl acetate:hexane/:3 containing 2% triethylamine as an eluent toyield 0.14 g (64%) 5′-O-dimethoxytrityl-2′-O-t-butyidimethylsilyl-N²phenylacetyl-6-S-dinitrophenylguanosine-3′-(2-cyanoethylN,N-diisopropylphosphoramidite) 22 as a yellow foam.

Example 7

[0111] Synthesis of 6-methyl-uridine phosphoramidite

[0112] Referring to FIG. 14, the suspension of 6-methyl-uracil (2.77 g,21.96 mmol) in the mixture of hexamethyidisilazane (50 mL) and drypyridine (50 mL) was refluxed for three hours. The resulting clearsolution of trimethylsilyl derivative of 6-methyl uracyl was evaporatedto dryness and coevaporated 2 times with dry toluene to remove traces ofpyridine. To the solution of the resulting clear oil, in dryacetonitrile, 1-O-acetyl-2′,3′,5′-tri-O-benzoyl-b-D-ribose (10.1 g, 20mmol) was added and the reaction mixture was cooled to 0° C. To theabove stirred solution, trimethylsilyl trifluoromethanesulfonate (4.35mL, 24 mmol) was added dropwise and the reaction mixture was stirred for1.5 h at 0° C. and then 1h at room temperature. After that the reactionmixture was diluted with dichloromethane washed with saturated sodiumbicarbonate and brine. The organic layer was evaporated and the residuewas purified by flash chromatography on silica gel withethylacetate-hexane (2:1) mixture as an eluent to give 9.5 g (83%) ofthe compound 2 and 0.8 g of the corresponding N¹,N³-bis-derivative.

[0113] To the cooled (-10° C.) solution of the compound (4.2 g, 7.36mmol) in the mixture of pyridine (60 mL) and methanol (10 mL) ice-cooled2M aqueous solution of sodium hydroxide (16 mL) was added with constantstirring. The reaction mixture was stirred at -10° C. for additional 30minutes and then neutralized to pH 7 with Dowex 50 (Py⁺). The resin wasfiltered off and washed with a 200 mL mixture of H₂O—Pyridine (4:1). Thecombined “mother liquor” and the washings were evaporated to dryness anddried by multiple coevaporation with dry pyridine. The residue wasredissolved in dry pyridine and then mixed with dimethoxytrityl chloride(2.99 g, 8.03 mmol). The reaction mixture was left overnight at roomtemperature. Reaction was quenched with methanol (25 mL) and the mixturewas evaporated. The residue was dissolved in dichloromethane, washedwith saturated aqueous sodium bicarbonate and brine. The organic layerwas dried over sodium sulfate and evaporated. The residue was purifiedby flash chromatography on silica gel using linear gradient of MeOH (2%to 5%) in CH₂Cl₂ as eluent to give 3.4 g (83%) of the compound 6.

Example 8

[0114] Synthesis of 6-methyl-cytidine phosphoramidite

[0115] Triethylamine (13.4 ml, 100 mmol) was added dropwise to a stirredice-cooled mixture of 1,2,4-triazole (6.22g, 90 mmol) and phosphorousoxychloride (1.89 ml, 20 mmol) in 50 ml of anhydrous acetonitrile. Tothe resulting suspension the solution of 2′,3′,5′-tri-O-Benzoyl-6-methyluridine (5.7 g, 10 mmol) in 30 ml of acetonitrile was added dropwise andthe reaction mixture was stirred for 4 hours at room temperature. Thenit was concentrated in vacuo to minimal volume (not to dryness). Theresidue was dissolved in chloroform and washed with water, saturated aqsodium bicarbonate and brine. The organic layer was dried over sodiumsulfate and the solvent was removed in vacuo. The residue was dissolvedin 100 ml of 1,4-dioxane and treated with 50 mL of 29% aq NH₄OHovernight. The solvents were removed in vacuo. The residue was dissolvedin the in the mixture of pyridine (60 mL) and methanol (10 mL), cooledto -15° C. and ice-cooled 2M aq solution of sodium hydroxide was addedunder stirring. The reaction mixture was stirred at -10 to -15° C. foradditional 30 minutes and then neutralized to pH 7 with Dowex 50 (Py⁺).The resin was filtered off and washed with 200 mL of the mixture H₂O—Py(4:1). The combined mother liquor and washings were evaporated todryness. The residue was cristallized from aq methanol to give 1.6 g(62%) of 6-methyl cytidine.

[0116] To the solution of 6-methyl cytidine (1.4 g, 5.44 mmol) in drypyridine 3.11 mL of trimethylchlorosilane was added and the reactionmixture was stirred for 2 hours at room temperature. Then aceticanhydride (0.51 mL, 5.44 mmol) was added and the reaction mixture wasstirred for additional 3 hours at room temperature. TLC showeddisappearance of the starting material and the reaction was quenchedwith MeOH (20 mL), ice-cooled and treated with water (20 mL, 1 hour).The solvents wee removed in vacuo and the residue was dried by fourcoevaporations with dry pyridine. Finally it was redissolved in drypyridine and dimethoxytrityl chloride (2.2 g, 6.52 mmol) was added. Thereaction mixture was stirred overnight at room temperature and quenchedwith MeOH (20 mL). The solvents were removed in vacuo. The remaining oilwas dissolved in methylene chloride, washed with saturated sodiumbicarbonate and brine. The organic layer was separated and evaporatedand the residue was purified by flash chromatography on silica gel withthe gradient of MeOH in methylene chloride (3% to 5%) to give 2.4 g(74%) of the compound (4).

Example 9

[0117] Synthesis of 6-aza-uridine and 6-aza-cytidine

[0118] To the solution of 6-aza uridine (5 g, 20.39 mmol) in drypyridine dimethoxytrityl chloride (8.29 g, 24.47 mmol) was added and thereaction mixture was left overnight at room temperature. Then it wasquenched with methanol (50 mL) and the solvents were removed in vacuo.The remaining oil was dissolved in methylene chloride and washed withsaturated aq sodium bicarbonate and brine. The organic layer wasseparated and evaporated to dryness. The residue was additionally driedby multiple coevaporations with dry pyridine and finally dissolved indry pyridine. Acetic anhydride (4.43 mL, 46.7 mmol) was added to theabove solution and the reaction mixture was left for 3 hours at roomtemperature. Then it was quenched with methanol and worked-up as above.The residue was purified by flash chromatography on silics gel usingmixture of 2% of MeOH in methylene chloride as an eluent to give 9.6 g(75%) of the compound.

[0119] Triethylamine (23.7 ml, 170.4 mmol) was added dropwise to astirred ice-cooled mixture of 1,2,4-triazole (10.6 g, 153.36 mmol) andphosphorous oxychloride (3.22 ml, 34.08 mmol) in 100 ml of anhydrousacetonitrile. To the resulting suspension the solution of2′,3′-di-O-Acetyl-5′-O-Dimethoxytrityl-6-aza Uridine (7.13 g, 11.36mmol) in 40 ml of acetonitrile was added dropwise and the reactionmixture was stirred for 6 hours at room temperature. Then it wasconcentrated in vacuo to minimal volume (not to dryness). The residuewas dissolved in chloroform and washed with water, saturated aq sodiumbicarbonate and brine. The organic layer was dried over sodium sulfateand the solvent was removed in vacuo. The residue was dissolved in 150ml of 1,4-dioxane and treated with 50 mL of 29% aq NH₄OH for 20 hours atroom temperature. The solvents were removed in vacuo. The residue waspurified by flash chromatigraphy on silica gel using linear gradient ofMeOH (4% to 10%) in methylene chloride as an eluent to give 3.lg (50%)of azacytidine.

[0120] To the stirred solution of 5′-O-Dimethoxytrityl-6-aza cytidine (3g, 5.53 mmol) in anhydrous pyridine trimethylchloro silane (2.41 mL, 19mmol) was added and the reaction mixture was left for 4 hours at roomtemperature. Then acetic anhydride (0.63 mL, 6.64 mmol) was added andthe reaction mixture was stirred for additional 3 hours at roomtemperature. After that it was quenched with MeOH (15 mL) and thesolvents were removed in vacuo. The residue was treated with 1M solutionof tetrabutylammonium fluoride in THF (20°, 30 min) and evaporated todryness. The remaining oil was dissolved in methylene chloride, washedwith saturated aq sodium bicarbonate and water. The separated organiclayer aws dried over sodium sulfate and evaporated to dryness. Theresidue was purified by flash chromatography on silica gel using 4% MeOHin methylene chloride as an eluent to give 2.9 g (89.8%) of thecompound.

[0121] General Procedure for the Introducing of the TBDMS-Group: To thestirred solution of the protected nucleoside in 50 mL of dry THF andpyridine (4 eq) AgNO₃ (2.4 eq) was added. After 10 minutestert-butyidimethylsilyl chloride (1.5 eq) was added and the reactionmixture was stirred at room temperature for 12 hours. The resultedsuspension was filtered into 100 mL of 5% aq NaHCO₃. The solution wasextracted with dichloromethane (2x100 mL). The combined organic layerwas washed with brine, dried over Na₂SO₄ and evaporated. The residue waspurified by flash chromatography on silica gel with hexanes-ethylacetate(3:2) mixture as eluent.

[0122] General Procedure for Phosphitylation: To the ice-cooled stirredsolution of protected nucleoside (1 mmol) in dry dichloromethane (20 mL)under argon blanket was added dropwise via syringe the premixed solutionof N,N-diisopropylethylamine (2.5eq) and 2-cyanoethylN′N-diisopropylchlorophosphoramidite (1.2 eq) in dichloromethane (3 mL).Simultaneously via another syringe N-methylimidazole (1 eq) was addedand stirring was continued for 2 hours at room tenperature. After thatthe reaction mixture was again ice-cooled and quenched with 15 ml of drymethanol. After 5 min stirring, the mixture was concentrated in vacuo(<40° C.) and purified by flash chromatography on silica gel usinghexanes-ethylacetate mixture contained 1% triethylamine as an eluent togive corresponding phosphoroamidite as white foam.

Example 10

[0123] RNA cleavage activity of HHA ribozyme substituted with6-methyl-Uridine

[0124] Hammerhead ribozymes targeted to site A (see FIG. 15) weresynthesized using solid-phase synthesis, as described above. U4 positionwas modified with 6-methyl-uridine.

[0125] RNA cleavage assay in vitro:

[0126] Substrate RNA is 5′ end-labeled using [γ-³²P] ATP and T4polynucleotide kinase (US Biochemicals). Cleavage reactions were carriedout under ribozyme “excess” conditions. Trace amount (≦1 nM) of 5′end-labeled substrate and 40 nM unlabeled ribozyme are denatured andrenatured separately by heating to 90° C. for 2 min and snap-cooling onice for 10 -15 min. The ribozyme and substrate are incubated,separately, at 37° C. for 10 min in a buffer containing 50 mM Tris-HCland 10 mM MgCl₂. The reaction is initiated by mixing the ribozyme andsubstrate solutions and incubating at 37° C. Aliquots of 5 μl are takenat regular intervals of time and the reaction is quenched by mixing withequal volume of 2X formamide stop mix. The samples are resolved on 20 %denaturing polyacrylamide gels. The results are quantified andpercentage of target RNA cleaved is plotted as a function of time.

[0127] Referring to FIG. 16, hammerhead ribozymes containing6-methyl-uridine modification at U4 position cleave the target RNAefficiently.

Example 11

[0128] RNA cleavage activity of HHB ribozyme substituted with6-methyl-Uridine

[0129] Hammerhead ribozymes targeted to site B (see FIG. 17) weresynthesized using solid-phase synthesis, as described above. U4 and U7positions were modified with 6-methyl-uridine.

[0130] RNA cleavage reactions were carried out as described above.Referring to FIG. 18, hammerhead ribozymes containing 6-methyl-uridinemodification at U4 and U7 positions cleave the target RNA efficiently.

Example 12

[0131] RNA cleavage activity of HHC ribozyme substituted with6-methyl-Uridine

[0132] Hammerhead ribozymes targeted to site C (see FIG. 19) weresynthesized using solid-phase synthesis, as described above. U4 and U7positions were modified with 6-methyl-uridine.

[0133] RNA cleavage reactions were carried out as described above.Referring to FIG. 20, hammerhead ribozymes containing 6-methyl-uridinemodification at U4 positions cleave the target RNA efficiently.

[0134] Sequences listed in FIGS. 7, 15, 17 19 and the modificationsdescribed in these figures are meant to be non-limiting examples. Thoseskilled in the art will recognize that variants (base-substitutions,deletions, insertions, mutations, chemical modifications) of theribozyme and RNA containing other 2′-hydroxyl group modifications,including but not limited to amino acids, peptides and cholesterol, canbe readily generated using techniques known in the art, and are withinthe scope of the present invention.

Example 13

[0135] Inhibition of Rat smooth muscle cell proliferation by 6-methyl-Usubstituted ribozyme HHA.

[0136] Hammerhead ribozyme (HHA) is targeted to a unique site (site A)within c-myb mRNA. Expression of c-myb protein has been shown to beessential for the proliferation of rat smooth muscle cell (Brown et al.,1992 J. Biol. Chem. 267, 4625).

[0137] The ribozymes that cleaved site A within c-myb RNA describedabove were assayed for their effect on smooth muscle cell proliferation.Rat vascular smooth muscle cells were isolated and cultured as described(Stinchcomb et al., supra). HHA ribozymes were complexed with lipids anddelivered into rat smooth muscle cells. Serum-starved cells werestimulated as described by Stinchcomb et al., supra. Briefly,serum-starved smooth muscle cells were washed twice with PBS, and theRNA/lipid complex was added. The plates were incubated for 4 hours at37° C. The medium was then removed and DMEM containing 10% FBS,additives and 10 μM bromodeoxyuridine (BrdU) was added. In some wells,FBS was omitted to determine the baseline of unstimulated proliferation.The plates were incubated at 37° C. for 20-24 hours, fixed with 0.3%H₂O₂ in 100% methanol, and stained for BrdU incorporation by standardmethods. In this procedure, cells that have proliferated andincorporated BrdU stain brown; non-proliferating cells arecounter-stained a light purple. Both BrdU positive and BrdU negativecells were counted under the microscope. 300-600 total cells per wellwere counted. In the following experiments, the percentage of the totalcells that have incorporated BrdU (% cell proliferation) is presented.Errors represent the range of duplicate wells. Percent inhibition thenis calculated from the % cell proliferation values as follows: %inhibition=100-100 (Ribozyme-0% serum)/(Control-0% serum).

[0138] Referring to FIG. 21, active ribozymes substituted with6-methyl-U at position 4 of HHA were successful in inhibiting rat smoothmuscle cell proliferation. A catalytically inactive ribozyme (inactiveHHA), which has two base substitutions within the core (these mutationsinactivate a hammerhead ribozyme; Stinchcomb et al., supra), does notsignificantly inhibit rat smooth muscle cell proliferation.

Example 14

[0139] Inhibition of stromelysin production in human synovial fibroblastcells by 6-methyl-U substituted ribozyme HHC.

[0140] Hammerhead ribozyme (HHC) is targeted to a unique site (site C)within stromelysin mRNA.

[0141] The general assay was as described (Draper et al., supra).Briefly, fibroblasts, which produce stromelysin, are serum-starvedovernight and ribozymes or controls are offered to the cells the nextday. Cells were maintained in serum-free media. The ribozyme wereapplied to the cells as free ribozyme, or in association with variousdelivery vehicles such as cationic lipids (including Transfectam™,Lipofectin™ and Lipofectamine™), conventional liposomes,non-phospholipid liposomes or biodegradable polymers. At the time ofribozyme addition, or up to 3 hours later, lnterleukin-1α (typically 20units/ml) can be added to the cells to induce a large increase instromelysin expression. The production of stromelysin can then bemonitored over a time course, usually up to 24 hours.

[0142] Supernatants were harvested 16 hours after IL-1 induction andassayed for stromelysin expression by ELISA. Polyclonal antibody againstMatrix Metalloproteinase 3 (Biogenesis, NH) was used as the detectingantibody and anti-stromelysin monoclonal antibody was used as thecapturing antibody in the sandwich ELISA (Maniatis et al., supra) tomeasure stromelysin expression.

[0143] Referring to FIG. 22, HHC ribozyme containing 6-methyl-Umodification, caused a significant reduction in the level of stromelysinprotein production. Catalytically inactive HHC had no significant effecton the protein level.

[0144] Other embodiments are within the following claims. TABLE ICharacteristics of Ribozymes Group I Introns Size: ˜200 to >1000nucleotides. Requires a U in the target sequence immediately 5′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of cleavage site. Over75 known members of this class. Found in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. RNAseP RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portionof a ribonucleoprotein enzyme. Cleaves tRNA precursors to form maturetRNA. Roughly 10 known members of this group all are bacterial inorigin. Hammerhead Ribozyme Size: ˜13 to 40 nucleotides. Requires thetarget sequence UH immediately 5′ of the cleavage site. Binds a variablenumber nucleotides on both sides of the cleavage site. 14 known membersof this class. Found in a number of plant pathogens (virusoids) that useRNA as the infectious agent (FIG. 1) Hairpin Ribozyme Size: ˜50nucleotides. Requires the target sequence GUC immediately 3′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of the cleavage site anda variable number to the 3′ side of the cleavage site. Only 3 knownmember of this class. Found in three plant pathogen (satellite RNAs ofthe tobacco ringspot virus, arabis mosaic virus and chicory yellowmottle virus) which uses RNA as the infectious agent (FIG. 3). HepatitisDelta Virus (HDV) Ribozyme Size: 50-60 nucleotides (at present).Cleavage of target RNAs recently demonstrated. Sequence requirements notfully determined. Binding sites and structural requirements not fullydetermined, although no sequences 5′ of cleavage site are required. Only1 known member of this class. Found in human HDV (FIG. 4). Neurospora VSRNA Ribozyme Size: ˜144 nucleotides (at present) Cleavage of target RNAsrecently demonstrated. Sequence requirements not fully determined.Binding sites and structural requirements not fully determined. Only 1known member of this class. Found in Neurospora VS RNA (FIG. 5).

1. Method for production of a modified ribozyme having a higherbiological activity compared to an unmodified ribozyme recognizing thesame substrate as said modified ribozyme, said modified and unmodifiedribozyme having identical enzymatic portions, comprising the step offorming said modified ribozyme with one or more modified bases in asubstrate binding arm of said modified ribozyme.
 2. The method of claim1 , wherein said modified base has a modification in the chemicalstructure of a nucleotide base selected from adenosine, guanosine,cytosine or uracil which has an effect on the ability of that base tohydrogen bond with its normal complementary base, either by increasingor decreasing the strength of the hydrogen bonding.
 3. The method ofclaim 1 , wherein said modified ribozyme has the structure of ahammerhead, hairpin, VS RNA or hepatitis delta virus derived ribozyme.4. The method of claim 3 , wherein said hammerhead ribozyme consists of32 to 40 nucleotide bases.
 5. The method of claim 1 , wherein saidmodified ribozyme and unmodified ribozyme have different bindingfree-energies with a target RNA.
 6. The method of claim 1 , wherein saidmodified base is selected from one shown in FIGS. 6a-d or FIG. 8 or FIG.14.
 7. Enzymatic nucleic acid having a non-natural base which enhancesthe enzymatic activity of said nucleic acid compared to an identicalnucleic acid having a naturally-occurring base in place of saidnon-natural base.
 8. The enzymatic nucleic acid of claim 7 , whereinsaid non-natural base is selected from one shown in FIGS. 6a-d or FIG. 8or FIG.
 14. 9. The enzymatic nucleic acid of claim 7 , wherein saidnucleic acid has a hammerhead motif.
 10. An enzymatic nucleic acidhaving atleast one 6-methyl-uridine substitution.
 11. A mammalian cellcomprising an enzymatic nucleic acid molecule of any one of the claims1-9.
 12. Hammerhead ribozyme having a modified base at position 4 or 7.13. The ribozyme of claim 12 , wherein said base is 6-methyl uridine.